Journal
ACS NANO
Volume 13, Issue 1, Pages 633-641Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acsnano.8b07697
Keywords
nanopore enzymology; phosphorylation; protein kinases; single molecule; kinase mechanism; metal ions
Categories
Funding
- National Institutes of Health
- Oxford Nanopore Technologies
- Biotechnology and Biological Sciences Research Council
- Structural Genomics Consortium from AbbVie [1097737]
- Bayer Pharma AG
- Boehringer Ingelheim
- Canada Foundation for Innovation
- Eshelman Institute for Innovation
- Genome Canada
- Innovative Medicines Initiative (EU/EFPIA) (ULTRA-DD Grant) [115766]
- Janssen
- Merck KGaA Darmstadt Germany
- MSD
- Novartis Pharma AG
- Ontario Ministry of Economic Development and Innovation
- Pfizer
- Sao Paulo Research Foundation-FAPESP
- Takeda
- Wellcome [106169/ZZ14/Z]
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Reversible protein phosphorylation plays a crucial and ubiquitous role in the control of almost all cellular processes. The interplay of protein kinases and phosphatases acting in opposition ensures tight dynamic control of protein phosphorylation states within the cell. Previously, engineered alpha-hemolysin pores bearing kinase substrate peptides have been developed as single-molecule stochastic sensors for protein kinases. Here, we have used these pores to observe, label-free, the phosphorylation and dephosphorylation of a single substrate molecule. Further, we investigated the effect of Mg2+ and Mn2+ upon substrate and product binding and found that Mn2+ relaxes active-site specificity toward nucleotides and enhances product binding. In doing so, we demonstrate the power and versatility of nanopore enzymology to scrutinize a critical post-translational modification.
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